Metabolic heart disease (MHD) is a common cardiomyopathy characterized by impaired mitochondrial energetics and hemodynamic dysfunction for which there are no specific therapies. The mechanism responsible for impaired myocardial energetics and hemodynamic dysfunction in MHD is not known. We found that sarcoplasmic reticulum Ca++-ATPase (SERCA) knock-in (SKI) mice in which C674 is replaced by a redox- insensitive serine are protected from MHD caused by HFHS-feeding. This finding reveals a knowledge gap in our understanding of MHD, as current models do not explain how oxidation of SERCA leads to the development of MHD. Emerging evidence suggests that Ca++ plays a key role in regulating mitochondrial function, and conversely, that limited mitochondrial Ca++ may decrease ATP generation and increase ROS production. Impaired mitochondrial Ca++ signaling may reflect decreased sarcoplasmic reticulum (SR) stores due to decreased refilling via SERCA and/or leak via the ryanodine receptor. In addition, elevated cytosolic [Na]i due to decreased Na+/K+-ATPase activity and/or increased sarcolemmal Na+/Ca++ exchange may impair mitochondrial Ca++ signaling by increasing mitochondrial Ca++ efflux via the mitochondrial Na+/Ca++ exchanger (NCXmito). These observations lead to our central hypothesis that oxidative inhibition of SERCA impairs mitochondrial Ca++ signaling, thereby leading to decreased ATP generation and increased ROS production that play key roles in the pathogenesis of MHD. Using HFHS-fed mice as a model of MHD we will pursue 3 interrelated aims to test the working hypothesis that interventions that correct mitochondrial Ca++ signaling by a) preventing the oxidation of SERCA, b) mitigating the effects of elevated cytosolic [Na+]i or c) scavenging mitochondrial ROS will improve myocardial energetics and hemodynamic function in MHD. In Aim 1 we examine the hypothesis that inhibition of SERCA due to thiol oxidation contributes to impaired mitochondrial Ca++ signaling that causes energetic dysfunction and excess ROS production in MHD. In Aim 2 we examine the hypothesis that elevated cytosolic [Na+]i impairs mitochondrial Ca++ signaling thereby contributing to energetic dysfunction and excess ROS production in MHD. In Aim 3 we examine the hypothesis that interventions from Aims 1 and 2 that correct mitochondrial Ca++ signaling will decrease a) mitochondrial ROS production and b) the related protein thiol oxidation in HFHS-fed mice. Innovative methods were developed to address these aims. We will assess energetics by 31P NMR and hemodynamic function simultaneously in beating hearts, and measure mitochondrial Ca++ and ROS in intact myocytes using genetically-targeted indicators delivered in vivo. In Aim 3 we use Tandem Mass Tags combined with multiple reaction monitoring MS to assess the cumulative effects of ROS on thiol oxidation targets and differentiate the effects of interventions from all 3 aims. The contribution of the proposed research would be significant, in our opinion, by resulting in new mechanistic understanding and new approaches to the prevention and treatment of MHD.